The invention relates to methods and devices to rapidly correlate individual cell morphology and cell surface characteristics with the same individual cell's cytoplasm or nuclear biochemical constituents, and to perform this analysis over an entire cell population.
Living cells are extremely complex entities that represent the fundamental building blocks of higher forms of life. Accordingly, there is great interest in observing and analyzing cells. Many methods and related devices exist that allow researchers to perform experiments on cells, both living and dead, and to observe such cells, but given the complexity of the problem, further advances are desirable.
All eukaryotic cells (with the exception of red calls and platelets) contain a nucleus (which contains the bulk of the cell's genetic material) surrounded by cytoplasm. In the cytoplasm, enzymes and RNA, controlled by the cell's genetic material, conduct thousands of specialized biochemical reactions. Some of these biochemical reactions are common to all cells, but others are unique to the particular cell type in question. The cell cytoplasm is in turn covered by a cell membrane, which itself is usually quite complex. Typically, cell membranes consist of hundreds or thousands of different types of membrane proteins and specialized lipids, embedded in a fluid two-dimensional lipid bilayer. Just as the cytoplasm has different biochemical pathways that differ according to cell type and function, so the cell membrane has different membrane proteins and lipids that also differ according to the cell type and function.
The composition of the cell's various membrane receptor and transporter molecules, and the biochemical pathways and constituents in the cell's cytoplasm, usually control the cell's morphology (size, shape, and structure as seen under a microscope). As one example, red cell cytoplasm contains various biochemical pathways to produce adenosine triphosphate (ATP). This cytoplasmic ATP, in turn, provides power to the red cell's cytoskeletal membrane components and ion transport molecules, which these membrane components use to maintain the red cell's shape and size.
As red cells age or become damaged, their ATP levels usually drop, and the red cell's morphology (shape and size changes). Thus a correlation often exists between a red cell's particular cell surface morphology and its cytoplasmic ATP levels.
As a second example, immune system B-cells, which produce antibodies, typically have both B-cell specific antigen detecting receptor molecules on their cell surfaces, and B-cell specific biochemical pathways (some of which act to produce antibodies) going on in the cytoplasm. B-cell specific genes that are activated in the B-cell nucleus in turn control these B-cell specific membrane components and cytoplasmic biochemical pathways.
Similarly, immune system T cells, which don't produce antibodies, but which play an active role in immune system regulation, as well as detecting and destroying pathogens, have a different set of membrane receptors, different cytoplasmic biochemical pathways and components, and different genes that are activated in the nucleus. It is likely that each of the many thousands or millions of different cell types in the body has its own unique pattern of morphology, membrane molecules, cytoplasmic molecular pathways, and nuclear genetic activation states.
Many of the advances in modern medicine and pharmacology rest upon fundamental research that has studied correlations between the cell morphology, membrane molecules, cytoplasmic molecules, and the genetic activation pathways that exist between cell populations. As a result, there is much interest in analytical technology that can enable researchers to better understand these correlations.
Although there have been many advances in analytical technology in these areas in recent years, there is room for further improvement. At present, available analytical technology primarily allows researchers to largely study isolated parts of these various systems, rather than see all the interactions between these systems as they operate across entire cell populations.
As an example, microscopy methods allow researchers to study cell morphology. Cytological stains allow researchers to draw some inferences between, for example, cell morphology and cell membrane receptors, but the bulk of the present microscopy methods primarily work with dead and chemically processed cells, rather than living cell populations, and thus are prone to artifacts and distortion.
Biochemical methods allow researchers to grind up large numbers of cells, and study the biochemical pathways inside the large number of cells, but this process generally requires large amounts of material. As a result, traditional biochemical analysis tends to miss (average out) cell-to-cell differences, and is also prone to distortion because the greater the time interval is between disrupting the cell and analyzing the cell's cytoplasmic biochemical pathways, the greater the chance is that these pathways will become damaged or distorted.
Recently, cell-sorting techniques have become popular. Cell sorting techniques allow researchers to correlate cell morphology with various types of cell membrane molecules on a population basis, using intact living cells. These methods, exemplified by fluorescence-activated cell sorting (FACS) techniques, have greatly facilitated modern medicine, particularly in the field of cellular immunology.
Although FACS techniques represent a big step forward in allowing researchers to understand cellular properties on a population basis, these methods still do not allow researchers and clinicians to easily monitor the correlations that exist between the cell's morphology, the cell's surface molecules, and the complex biochemical pathways that occur inside of the cytoplasm or nucleus of these cells.
Earlier researchers realized that it would be desirable to produce devices that can, on a cell population basis, correlate individual cell morphology and surface characteristics with the cell's internal biochemistry. However, in spite of the long felt need for this type of device, no such device has yet been commercialized. This appears to be because prior art in this area did not perform well enough to produce robust and capable devices that would actually perform well in the hands of users, on a routine basis.
Prior art methods include U.S. Pat. No. 6,586,253 B1 to Harrison et. al.; U.S. Pat. No. 6,783,657 to Culberson et. al., US patent application 20040058423 to Albritton; and other methods.
Harrison teaches a microchip method for detecting cell contents, in which a cell is put into a fluid filled channel in a microchip, and diverted to a cell lytic region. There, the cells are lysed, and the cell contents are then analyzed at a detection zone, usually by fluorescence or luminescence detection means. Harrison's methods have not been commercialized, however, possibly because the invention did not teach any means to prevent cells or cell debris from fouling the apparatus in operation. Additionally Harrison failed to teach ways in which the analyzer might sort or screen particular cell populations prior to analysis, ways to correlate cell surface markers with cellular contents, or ways to correct the assay for distortions caused by inadequate lysis or variations in the volume of the cytoplasmic debris field of the lysed cells.
Culbertson teaches an alternate microfluidic cell analysis system. Similar to Harrison, Culberson introduces cells into a microfluidic chamber, and also utilizes electrical cell lytic techniques. However Culberson does not incorporate any cell morphology or cell surface analytical means in his device, and does not disclose means by which cell morphology or cell surface characteristics may be correlated with internal cell biochemical molecules. Like Harrison, Culberson also remains silent on methods to prevent his apparatus from becoming fouled by cells and cell debris.
Albritton teaches a “single-cell at a time” type method in which cells are lysed in a larger cell collection chamber, and the cell contents are immediately sucked into analytical capillary electrophoresis tubes. There, the cells are mixed with suitable reagents for analyzing the cytoplasm, and subjected to capillary electrophoresis. Although this method allows for very precise determination of certain cell contents, the methods are single cell based, rather than population based, and are generally unsuited for the high volume cell analysis methods contemplated by the invention.
In order to produce practical devices that can actually be used on a routine basis to solve research and clinical problems, issues such as cell pre-screening, device fouling, correction for dilution effects, correction for analytical artifacts, reasonable throughput, and adequate analytical capability need to be addressed. Here prior art has been silent.
Ideally, what is needed is some sort of rapid cell sorting or analysis device that can analyze a large population of cells (for example, the population of lymphocytes from a blood sample), and provide clinicians with accurate and timely cell population data that correlates the cell morphology (that is cell size, shape, or visual characteristics) and cell surface molecules on the surface of the various cells in this cell population with the biochemical and genetic pathways ongoing in the various cell's cytoplasm and nuclei. A device and method that would be capable of doing this correlation on an individual cell basis, and that would be capable of analyzing an entire cell population in this manner, would achieve commercial success, and would likely make many contributions to medical research as well. As will be seen, the invention provides such a device and method in an elegant manner.